ANALYSIS OF THE CHROMATIN-REMODELING COMPLEX RSC

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DESCRIPTION (provided by applicant): SWI/SNF-family complexes are large, abundant protein machines that mobilize nucleosomes to regulate the access of DNA-binding factors to chromatin. These complexes have a set of seven 'core' proteins that are conserved throughout eukaryotes, as well as additional proteins that tailor the complex for specialized functions. SWI/SNF-family chromatin remodeling complexes (termed 'remodelers') help regulate transcription, DNA repair and genome stability, among other processes. Beyond these critical roles in normal chromosomal biology, the misregulation of SWI/SNF remodelers is associated with many cancers, and is the cause of rhabdoid tumors. We focus on the SWI/SNF-family remodeler RSC, which is an essential and abundant remodeler from the budding yeast that has served as a prototype for understanding remodeler functions. RSC is a 15-protein complex that both slides and ejects nucleosomes. Our work addresses central issues in chromatin remodeling: how do remodelers reposition and eject nucleosomes, how are these remodeling modes regulated, and how do covalent modifications on nucleosomes affect RSC targeting and mechanism? RSC can be considered a mosaic of protein modules that work together to conduct chromatin remodeling: 1) a catalytic module that conducts DNA translocation, which involves an ATPase (Sth1) and a pair of actin-related proteins (ARPs), 2) a set of proteins with nucleosome-binding motifs (bromodomains) that may help target RSC to particular nucleosomes, and 3) a set of fungalspecific proteins that regulate RSC participation in particular chromosomal processes. Aim 1 applies biochemical, single-molecule, and structural approaches to understand how the DNA translocation module mobilizes and ejects nucleosomes, and also examines the structural engineering of the RSC-nucleosome complex. Aim 2 focuses on the regulation of the Sth1 ATPase by the ARPs and by three domains present on Sth1 (HSA, post-HSA, and Protrusion #1) which help link the ARPs to ATPase regulation. Aim 3 focuses on understanding how the eight bromodomains in RSC, especially the tandem bromodomains in the essential protein Rsc4, are involved in RSC targeting or activity. Insights from RSC should inform the functions of these similar proteins in mammalian SWI/SNF complexes, and contribute considerably to our understanding of the chromatin remodeling process.